Lysosomal Lipid Radical Probe -Lyso-NBD-Pen-L271 Product Manual

  Ferroptosis is a form of cell death caused by the abnormal accumulation of lipid peroxides in an iron‑dependent manner. During the progression of lipid peroxidation, various reaction intermediates are generated as a result of lipid oxidation. A recent report indicates that ferroptosis is initiated by lipid peroxidation of the lysosomal membrane, highlighting the importance of detecting lipid radicals within lysosomes¹⁾.
  Lyso‑NBD‑Pen is a fluorescent probe that localizes to lysosomes and selectively reacts with lipid radicals. When added to cultured cells, Lyso‑NBD‑Pen permeates the cell membrane, accumulates in lysosomes, and emits a fluorescent signal upon reaction with lipid radicals, enabling the detection of lysosomal lipid radicals in living cells.

Lysosomal Lipid Radical Probe -Lyso-NBD-Pen-

2 nmol

 

 

Store in a cool and dark place.

• Fluorescence microscope, or flow cytometer
• Incubator (37℃)
• Micropipettes (100–1000 µl, 20–200 µl, 1–10 µl)
• Microtubes
• Medium or Hanks' Balanced Salt Solution (HBSS)

• Centrifuge the tube briefly before opening the cap because the contents may adhere to the tube wall or the inside of the cap.
• Please refer to Table 1 for suitable fluorescence wavelengths for each application.

 

　　　　　　                                          Table 1. Recommended filter settings for Lyso-NBD-Pen

Applications	Fluorescence microscope	Flow cytometer
Measurement wavelength	・Confocal microscope Ex/Em: 488/490–600 nm ・Fluorescence microscope GFP filter	FITC filter

 

 

 

Preparation of 100 µmol/l Lyso-NBD-Pen DMSO stock solution

Add 20 μl of DMSO to a tube containing 2 nmol of Lyso-NBD-Pen and dissolve by pipetting to prepare a 100 µmol/l Lyso-NBD-Pen DMSO stock solution.

• The 100 µmol/l Lyso-NBD-Pen DMSO stock solution is stable at −20℃ for 1 month.

 

Preparation of 100 nmol/l Lyso-NBD-Pen working solution

Dilute the 100 µmol/l Lyso-NBD-Pen DMSO stock solution 1:1000 in medium to prepare 100 nmol/l Lyso-NBD-Pen working solution.

• The working solution cannot be stored and must be prepared each day freshly.

• Refer to Table 2 for the amount of  working solution required by vessel type.

 

                    　　　　Table 2. Required amount of the working solution by vessel type

Vessel	35-mm dish	ibidi 8-well plate	96-well black plate (clear bottom)
Appropriate amount	2 ml	200 μl/well	100 μl/well

 

1. Seed cells in a vessel. Culture the cells at 37°C overnight in a 5% CO₂ incubator.
2. Discard the supernatant and wash the cells once with medium.
3. Discard the supernatant, add an appropriate volume of 100 nmol/l Lyso-NBD-Pen working solution to the vessel, and incubate at 37°C for 30 min in a 5% CO₂ incubator.
4. Discard the supernatant and wash the cells twice with medium.
5. Add medium containing ferroptosis inducers or inhibitors, and incubate the cells at 37°C in a 5% CO₂ incubator for an appropriate time.
6. Discard the supernatant and wash the cells twice with HBSS.
7. Observe the cells under a fluorescence microscope or measure fluorescence signals using a flow cytometer.

Detection of lysosomal lipid radicals in HT-1080 cells treated with RSL3 using a confocal laser microscope

1.  HT-1080 cells (3×10⁴ cells/well) in MEM (supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin) were seeded in an ibidi 8-well plate and incubated at 37°C overnight in a 5% CO₂ incubator.
2. After the supernatant was removed, the cells were washed once with medium. Then, 100 nmol/l Lyso-NBD-Pen working solution (200 µl/well) was added to the cells, and they were incubated at 37°C for 30 min in a 5% CO₂ incubator.
3. The supernatant was removed, and the cells were washed twice with medium.
4. RSL3 (1 µmol/l) dissolved in medium was added to the cells, and they were incubated at 37°C for 2 h in a 5% CO₂ incubator.
5. The supernatant was removed, and the cells were washed twice with HBSS. The wells were refilled with HBSS.
6. The cells were observed under a confocal laser microscope.

Detection: Confocal laser microscope Ex/Em: 488/490–600 nm Scale bar: 20 µm

Figure 1. Fluorescence images of HT-1080 cells obtained with a confocal laser microscope

 

Detection of lysosomal lipid radicals in HT-1080 cells treated with RSL3 using a flow cytometer

1. HT-1080 cells (3×10⁵ cells/well) in MEM (supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin) were seeded in a 6-well plate and incubated at 37°C overnight in a 5% CO₂ incubator.
2. After the culture medium was removed, the cells were washed once with medium. Then, 100 nmol/l Lyso-NBD-Pen working solution (2 ml/well) was added to the cells, and they were incubated at 37°C for 30 min in a 5% CO₂ incubator.
3.  The supernatant was removed, and the cells were washed twice with medium.
4. RSL3 (1 µmol/l) dissolved in medium was added to the cells, and they were incubated at 37°C for 2 h in a 5% CO₂ incubator.
5. After removing the supernatant, the cells were washed once with HBSS and detached from the plate using 0.25% trypsin–EDTA.
6. The detached cells were collected into a 1.5‑ml tube containing serum-supplemented medium and centrifuged at 300 × g for 5 min.
7. The supernatant was discarded, 1 ml of PBS was added to resuspend the cells, and the cells were centrifuged at 300 × g for 5 min.
8. The supernatant was discarded, and the cells were resuspended in 1 ml of PBS.
9. The samples were passed through a cell strainer for flow cytometry and analyzed using a flow cytometer.

Detection: Flow cytometer FITC filter

Figure 2. Fluorescence signals from HT-1080 cells, detected using  a flow cytometer

 

1) K. Yamada et al., Nature Communications, 2025, 16, 2554.